CN116970650A - Envelope protein combination, targeting virus vector containing envelope protein combination and preparation method of targeting virus vector - Google Patents
Envelope protein combination, targeting virus vector containing envelope protein combination and preparation method of targeting virus vector Download PDFInfo
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Abstract
The invention discloses an envelope protein combination, a targeting virus vector containing the envelope protein combination and a preparation method. The envelope protein combination comprises RFV-F or a variant thereof and RFV-RBP or a variant thereof; the NCBI sequence number of the amino acid sequence of the RFV-F is NP-899659.1; the NCBI sequence number of the amino acid sequence of the RFV-RBP is NP-899660.1; variants of RFV-F are those in which a truncation of 1-80 amino acid residues occurs at RFV-F; a variant of RFV-RBP is a truncation of 1-80 amino acid residues occurring at the RFV-RBP. The envelope protein combination can effectively improve the transduction efficiency and the specificity of a delivery carrier tool, and can also carry out targeting modification according to different target cells.
Description
Technical Field
The invention relates to the technical field of biotechnology and biomedicine, in particular to an envelope protein combination, a targeting viral vector containing the envelope protein combination and a preparation method.
Background
Gene therapy is one of the most important technologies in basic life sciences and modern medicine, and aims to compensate or repair defective genes, and has been applied to the fields of hereditary monogenic diseases, cancers, and the like. However, an important watershed for gene therapy is whether the gene delivery pathway can be changed from ex vivo to in vivo, and how to achieve efficient and safe gene delivery in vivo remains a great challenge, which requires that the vector have high specificity for target cells to avoid off-target situations. In vivo delivery means that have been shown to be effective at present include viral vectors, lipid nanoparticles, and the like, and viral vectors are considered to be the most potential means due to their nucleic acid delivery capabilities naturally possessed by evolution, particularly Lentiviral (LV) vectors and adeno-associated virus (AAV) vectors, have been attracting attention in leading edge studies. Viral vectors require interaction with cellular receptors to trigger fusion of the viral envelope with the cell membrane when gene delivery is performed, a process typically mediated by viral envelope proteins to achieve cell tropism. Non-engineered AAV and LV vectors tend to accumulate in the liver and spleen when used in vivo and fail to reach target cells, and pseudotyped LV vectors with surface envelope proteins targeted to be equipped with specific antibodies are capable of gene delivery targeting specific cell populations depending on the antibody type. At the same time, CD34 in the circulatory system + Hematopoietic stem cells (hematopoietic stem cells, HSCs) are ideal target cells in gene therapy, as any genetic modification will be transferred to all lineages derived from them, which is important in the treatment of genetic monogenic diseases. Targeting human CD34 + Cells or other cell types such as CD105 + CellsLV vectors for gene delivery can effectively reduce the risk and cost of HSC gene therapy, and are of great significance in promoting the wide clinical application of gene therapy.
While the principle of this carrier is presently a public information, systematic optimization of the carrier is required to ensure that a more optimal delivery is achieved. None of these can be achieved and completed in a short time. (1) The German Paul-Ehrlich institute developed a pseudotyped lentiviral vector based on measles virus (MEV), an anti-CD 8-LV lentiviral vector targeting T lymphocytes for in vivo production of CD19-CAR T cells, the principle of the vector tool being to target T lymphocytes by a paramyxovirus envelope protein pseudotyped lentiviral vector equipped with an anti-CD 8 single chain antibody, thereby generating CAR T cells in vivo. (2) The university of california octocrylene developed a pseudotyped lentiviral vector based on nipah virus (NIV) that could increase its gene delivery efficiency in a variety of cell lines, but still did not possess cell specificity.
Delivery means currently used in vivo, including viral vectors (e.g., AAV, LV, etc.) and non-viral vectors (e.g., lipid nanoparticles, virus-like particles, etc.), encapsulate the substance to be delivered via a protein or lipid shell to protect them from degradation or sequestration prior to cell entry. Cell entry of AAV vectors is mediated by capsid proteins of a particular serotype, capsid engineering is typically achieved based on selection of capsids or insert peptides on capsids of different serotypes, which may mean that AAV vectors cannot be engineered directionally according to different cell types. The cellular entry process of non-viral vectors is determined by the physicochemical properties of the surface particles, rather than the naturally evolving biological entry mechanisms, and therefore may not be as highly ordered as viral vectors. LV vectors can be pseudotyped with different viral envelope proteins to alter their tropism for different cells, with vesicular stomatitis virus glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G) being the most commonly used class, as it can help lentiviral vectors achieve efficient gene delivery in a variety of cell types. However, VSV-G pseudotyped LV vectors have a broad cell tropism and limited delivery efficiency in resting blood cells (e.g., HSC, T/B lymphocytes, etc.) limiting their in vivo applications. To address this issue, VSV-G may be replaced with other viral envelope proteins, such as those of measles virus (MEV), nipah virus (NIV) in Paramyxoviruses (PMV), and other types of heterologous viruses such as baboon endogenous retrovirus (baboon endogenous retrovirus, baEV). Paramyxovirus envelope proteins contain two glycoproteins required for entry into cells, fusion protein (F) and Receptor Binding Protein (RBP), and attachment of target molecules to the outer domain of RBP can significantly enhance targeting of pseudotyped LV vectors to specific cell types. The great engineering potential also allows them to be applied in targeting LV vector development, for example NIV pseudotyped LV vectors equipped with anti-CD 8/anti-CD 20 antibodies targeting T/B lymphocytes. Meanwhile, when the LV vector is applied to in vivo targeting, the immunogenicity of viral envelope proteins and in vivo natural neutralizing antibodies need to be considered, the difficulty can be avoided to a certain extent by selecting envelope proteins of viruses with host types greatly different from human beings, and the targeting lentiviral vector based on the reptile paramyxovirus envelope proteins is expected to improve the existing gene delivery tool and realize efficient and specific in vivo delivery aiming at specific cell groups.
Although gene therapy has solved to some extent the problem that conventional therapeutic approaches are currently clinically difficult to cure radically, existing gene delivery tools still face a series of new challenges: (1) The delivery vector is difficult to take into account the high efficiency and the specificity, so that the existing tool can only be used in vivo after the target cells are modified in vitro and then returned to the patient, the mode is complicated in steps and high in cost, and (2) the targeting transformation potential of the vector is insufficient, and the targeting transformation can not be carried out on specific cell populations (such as HSC). There is therefore a need for a method to increase the efficiency and specificity of gene delivery while reducing the cost of treatment, which can be somewhat of the problems currently faced by the present invention.
Disclosure of Invention
In order to solve the problem that the existing delivery vector for gene therapy is difficult to be compatible with high efficiency and specificityThe invention provides an envelope protein combination, a targeting viral vector containing the envelope protein combination and a preparation method thereof, which solve the problems of complicated steps and high cost and incapability of carrying out targeting reconstruction on specific cell populations. The envelope protein combination and the targeting lentiviral vector containing the same provided by the invention effectively improve the targeting of human CD34 + Hematopoietic stem cells or other cell types + The transduction efficiency and the specificity of the cells can be improved in a targeting way according to different target cells, so that a foundation is laid for in-vivo delivery in the future, the safety is improved, the treatment cost is greatly reduced, and the method has a high practical value.
In order to solve the above technical problems, the first aspect of the present invention provides an envelope protein combination comprising RFV-F or a variant thereof and RFV-RBP or a variant thereof; the NCBI sequence number of the amino acid sequence of the RFV-F is NP-899659.1; the NCBI sequence number of the amino acid sequence of the RFV-RBP is NP-899660.1; variants of RFV-F are those in which a truncation of 1-80 amino acid residues occurs at RFV-F; a variant of RFV-RBP is a truncation of 1-80 amino acid residues occurring at the RFV-RBP.
In some embodiments, the variant of RFV-F and the variant of RFV-RBP truncate a portion or all of the intracellular region, a portion or all of the transmembrane region, and/or a portion of the extracellular region, respectively.
In some preferred embodiments, the amino acid sequence of the variant of RFV-F is an amino acid sequence truncated by 13, 32, 56, or 80 amino acids at the C-terminus of the sequence NCBI sequence No. np_899659.1; the amino acid sequence of the variant of RFV-RBP is an amino acid sequence truncated by 21, 32, 60 or 80 amino acids after the N-terminal initial amino acid M of the amino acid sequence of NCBI sequence No. NP-899660.1.
In some preferred embodiments, the envelope protein combination comprises the amino acid sequence of NCBI sequence No. np_899659.1 and the amino acid sequence of NCBI sequence No. np_ 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence of NCBI sequence No. np_ 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 32 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
The envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 32 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 60 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence with NCBI sequence number NP 899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminal initial amino acid M of the amino acid sequence with NCBI sequence number NP 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 80 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 56 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 32 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
The envelope protein combination comprises an amino acid sequence of NCBI sequence No. NP-899659.1 and an amino acid sequence truncated by 60 amino acids after the N-terminal starting amino acid M of the amino acid sequence of NCBI sequence No. NP-899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 60 amino acids after the N-terminal starting amino acid M of the amino acid sequence of NCBI sequence No. np_ 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 80 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 56 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 80 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 56 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminal starting amino acid M of the amino acid sequence of NCBI sequence No. np_ 899660.1.
In some further preferred embodiments, the RFV-RBP or variant thereof further incorporates an scFv; for example, the scFv is scFv-CD34, scFv-CD105 or other targeting sequence.
In order to solve the above technical problem, a second aspect of the present invention provides an envelope protein plasmid, the envelope protein plasmid or plasmid combination comprising a nucleotide sequence encoding the envelope protein combination according to the first aspect of the present invention; wherein the nucleotide sequences of RFV-F or variants thereof and RFV-RBP or variants thereof are located on the same envelope plasmid or on two different envelope plasmids pF and pRBP, respectively.
In some preferred embodiments, the nucleotide sequence of RFV-F is the 4965-6758 region of NCBI sequence No. NC_ 005084.2; the nucleotide sequence of the RFV-RBP is 6762-8699 interval with NCBI sequence number NC_ 005084.2.
In some embodiments, the nucleotide sequence of the variant of RFV-F is a nucleotide sequence truncated 39, 96, 168 or 240 nucleotides at the 3' end of the sequence in the 4965-6758 interval of NCBI sequence No. NC 005084.2; the nucleotide sequence of the variant of RFV-RBP is truncated by 63, 96, 180 or 240 nucleotides after the 5' -terminal initiation codon ATG of the sequence of interval 6762-8699 of NCBI sequence No. NC_ 005084.2.
In some preferred embodiments, the nucleotide sequence encoding the combination of envelope proteins comprises the nucleotide sequence of the 4965-6758 interval of NCBI sequence No. nc_005084.2 and the nucleotide sequence of the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 39 nucleotides at the 3' end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a nucleotide sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence 3 'to the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 63 nucleotide truncated nucleotide sequence after the 5' start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 96 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
The nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 63 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 96 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 180 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises the nucleotide sequence of the 4965-6758 interval of NCBI sequence No. NC_005084.2 and the nucleotide sequence truncated by 63 nucleotides after the 5' -terminal start codon ATG of the sequence of the 6762-8699 interval of NCBI sequence No. NC_ 005084.2; or alternatively, the first and second heat exchangers may be,
The nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 240 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 168 nucleotides at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a nucleotide sequence truncated by 96 nucleotides after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises the nucleotide sequence of the 4965-6758 interval of NCBI sequence No. NC_005084.2 and the nucleotide sequence truncated 180 nucleotides after the 5' -terminal start codon ATG of the sequence of the 6762-8699 interval of NCBI sequence No. NC_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 180 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
The nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 240 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 168 nucleotides at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a nucleotide sequence truncated by 240 nucleotides after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 168 nucleotides at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. NC_005084.2 and a nucleotide sequence truncated by 63 nucleotides after the 5' end initiation codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. NC_ 005084.2.
In some preferred embodiments, the RFV-RBP or variant thereof further comprises an scFv fused thereto; for example, the nucleotide sequence of the scFv is a nucleotide sequence encoding scFv-CD34, scFv-CD105, or other targeting sequence.
In some embodiments, in the combination of envelope plasmids, the nucleotide sequences of the RFV-F or variant thereof and the RFV-RBP gene or variant thereof are located on two different envelope plasmids, respectively.
In some preferred embodiments, the envelope plasmids are pF and pRBP.
In some embodiments, the envelope plasmid further comprises the following genetic elements: cytomegalovirus enhancer, cytomegalovirus promoter, human β -globin intron, kozak consensus sequence, and/or human β -globin polyadenylation signal.
In some embodiments, the backbone vector used by the envelope plasmid is a pMD2.G vector.
In order to solve the above technical problem, a third aspect of the present invention provides a lentiviral vector packaging system comprising an envelope protein plasmid or a plasmid combination according to the second aspect of the present invention.
The targeted viral vector is capable of targeting specific cell populations, enabling efficient and specific gene delivery. The virus vector is designed based on a third-generation lentivirus vector, is specially modified for lentivirus envelope proteins, and can realize targeted gene delivery in different cell populations. The targeting viral vector used in the invention is a pseudo replication defective lentiviral vector, the original gene of the virus is completely knocked out, the virus is not pathogenic per se, and envelope proteins are replaced by fusion proteins (F) of the original vesicular stomatitis virus glycoprotein (VSV-G) and receptor binding proteins (RBP-scFv) provided with single chain antibodies of reptile paramyxovirus (Reptilian ferlavirus, RFV), which respectively exert the membrane fusion function and the receptor recognition function between the lentiviral vector and target cells, so that efficient and specific gene delivery aiming at specific cells is realized. The targeting virus vector is obtained by cotransfecting a packaging cell line with a third generation lentivirus helper packaging plasmid, a shuttle vector plasmid and an envelope protein plasmid, wherein the envelope protein plasmid mainly comprises the following elements (A of figure 2): cytomegalovirus enhancer (CMV enhancer), cytomegalovirus promoter (CMV promoter), human β -globin intron (β -globin intron), kozak consensus sequence (Kozak), envelope protein (Envelope glycoprotein) coding region sequence, human β -globin polyadenylation signal (β -globin poly a). One of the targeting viral vectors comprises two differently functioning envelope protein plasmids, a fusion protein (RFV-F) and a receptor binding protein equipped with a single chain antibody (RFV-RBP-scFv) (B of FIG. 2). To further optimize the vector, truncations of RFV-F and RFV-RBP-scFv were constructed and tested for titer and transduction effect in the present invention to improve the packaging and delivery capacity of the vector.
In some preferred embodiments, the lentiviral vector packaging system further comprises a helper packaging plasmid and/or a shuttle vector plasmid.
In some more preferred embodiments, the packaging plasmid, the shuttle vector plasmid, and the envelope plasmid or plasmid combination are present in a ratio of (3-1): 3-1, e.g., 1:1:1.
In some even more preferred embodiments, the ratio of the envelope plasmids pF and pRBP-scFv in the envelope plasmid combination is (1-5): 1, e.g., 3:1; the helper plasmid was psPAX2 and the shuttle plasmid was pCDH.
The targeting viral vectors of the application are capable of targeting specific cell populations in different cell populations, which vectors are less efficient for conventional lentiviral vector transduction of CD34 + Hematopoietic stem cells or other cell types + Cells can also accomplish efficient transduction. In addition, the vector developed based on the method can be used for directly targeting hematopoietic stem cells in vivo for gene delivery in the future, reduces risks introduced by external operations such as mobilization, marrow removal, infection and culture in the application process, can effectively reduce the risks and cost of hematopoietic stem gene therapy, and has important significance for promoting the wider clinical application of hematopoietic stem cell gene therapy, shortening the treatment process and reducing the treatment cost. In addition, the targeting viral vector of the present application can be applied to target cells or delivery of target genes or shRNA, influencing cell differentiation state, signaling pathway, etc.
In order to solve the above technical problem, the fourth aspect of the present invention provides a transformant transfected with the envelope protein plasmid or plasmid combination according to the second aspect of the present invention or the lentiviral vector packaging system according to the third aspect of the present invention.
In some preferred embodiments, the recipient cell of the transformant is an animal cell.
In some more preferred embodiments, the recipient cell of the transformant is HEK293T.
In order to solve the above technical problem, a fifth aspect of the present invention provides a targeting viral vector comprising the envelope protein combination according to the first aspect of the present invention.
In some preferred embodiments, the targeted viral vector is obtained by culturing a transformant according to the fourth aspect of the present invention.
In order to solve the technical problem, a sixth aspect of the present invention provides a method for preparing the targeting viral vector according to the fifth aspect of the present invention, and culturing the transformant according to the fourth aspect of the present invention to obtain the targeting viral vector.
In order to solve the technical problem described above, a seventh aspect of the present invention provides a pharmaceutical composition comprising the transformant according to the fourth aspect of the present invention or the targeted viral vector according to the fifth aspect of the present invention.
In order to solve the above technical problem, the eighth aspect of the present invention provides a kit comprising an envelope protein according to the first aspect of the present invention, an envelope protein plasmid or a plasmid combination according to the second aspect of the present invention, a lentiviral vector packaging system according to the third aspect of the present invention, a transformant according to the fourth aspect of the present invention, a targeted viral vector according to the fifth aspect of the present invention or a pharmaceutical composition according to the seventh aspect of the present invention.
In order to solve the above technical problems, the ninth aspect of the present invention provides a method for delivering a gene, ribonucleoprotein complex or drug by an envelope protein according to the first aspect of the present invention, an envelope protein plasmid or a plasmid combination according to the second aspect of the present invention, a lentiviral vector packaging system according to the third aspect of the present invention, a transformant according to the fourth aspect of the present invention, a targeted viral vector according to the fifth aspect of the present invention, a pharmaceutical composition according to the seventh aspect of the present invention or a kit according to the eighth aspect of the present invention.
In some preferred embodiments, the method is for non-therapeutic purposes.
In order to solve the technical problem, the tenth aspect of the present invention provides an application of the envelope protein of the first aspect of the present invention, the envelope protein plasmid or the plasmid combination of the second aspect of the present invention, the lentiviral vector packaging system of the third aspect of the present invention, the transformant of the fourth aspect of the present invention, the targeting viral vector of the fifth aspect of the present invention, the pharmaceutical composition of the seventh aspect of the present invention or the kit of the eighth aspect of the present invention in preparing a gene therapy drug.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: (1) Through the novel design of the targeting lentiviral vector, the transduction efficiency and the specificity of a delivery vector tool are effectively improved, and the in vivo delivery can greatly reduce the gene therapy cost; (2) The targeted lentiviral vector has great transformation potential, and the targeted delivery can be realized aiming at specific cell groups by replacing specific antibodies of the vector, so that the application range of the existing viral vector is further expanded. Lays a foundation for future in vivo delivery, greatly reduces the treatment cost while increasing the safety, and has great practical value.
Drawings
FIG. 1 is a screen for paramyxoviruses;
FIG. 2 is the construction of a targeted lentiviral vector;
FIG. 3 shows the structure of a plasmid of the envelope protein of the reptile paramyxovirus;
FIG. 4 is a graph showing transduction of different paramyxovirus envelope protein pseudotyped targeting viruses in target/non-target cells;
FIG. 5 is a scheme for truncating the RFV envelope protein;
FIG. 6 is a diagram showing transduction of a targeted virus pseudotyped with a truncate in a target cell;
FIG. 7 shows the transduction positive rate and viral titer of a truncating pseudotyped targeting virus in a target cell;
FIG. 8 shows GFP expression after RFV envelope truncating pseudotyped targeting virus transduced target/non-target cells (HT 1080-CD34/HT 1080);
FIG. 9 shows GFP expression after RFV envelope truncating pseudotyped targeting virus transduced target/non-target cells (HT 1080 (CD105+)/HT 1080 (CD 105-));
FIG. 10 is a transduction test of a mixed cell population of RFV envelope protein truncations pseudotyped transduced target cells and non-target cells.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
EXAMPLE 1 construction of recombinant lentiviral vector envelope protein plasmid
The recombinant lentiviral vector of this example was based on a third generation lentiviral vector, with pseudotyped replacement for the viral envelope protein.
(1) The envelope proteins are replaced by the original vesicular stomatitis virus glycoprotein (VSV-G) respectively by Fusion proteins (Fusion, F) of MEV, NIV, MUV, AMAV, SPV, RFV and BEIPV and Receptor Binding Proteins (RBP) provided with single-chain antibodies, and the sequence sources are shown in the table 1 so as to screen the paramyxovirus envelope proteins with good effects for subsequent experiments.
TABLE 1 Paramyxoviral envelope protein sequence Source list
(2) The envelope proteins were replaced by the original vesicular stomatitis virus glycoprotein (VSV-G) with the fusion protein of reptile paramyxovirus (RFV-F) and the receptor binding protein equipped with single chain antibodies (RFV-RBP-scFv), and were truncated to various extents with respect to their transmembrane regions, to improve virus packaging efficiency and transduction efficiency.
The RFV-F gene sequence adopted in this example is the 4965-6758 region with NCBI sequence No. NC_005084.2, the NCBI sequence No. NP-899659.1 with the amino acid sequence, the RFV-RBP gene sequence is the 6762-8699 region with NCBI sequence No. NC_005084.2, the amino acid sequence of the amino acid sequence is NP-899660.1, the amino acid sequences of RFV-Fd13, RFV-Fd32, RFV-Fd56, RFV-Fd80 are respectively 13, 32, 56 or 80 amino acids truncated at the C-terminal of the peptide chain of RFV-Fd0 (NCBI sequence No. NP-899659.1); the gene sequences of RFV-Fd13, RFV-Fd32, RFV-Fd56 and RFV-Fd80 are respectively truncated by 39, 96, 168 or 240 nucleotides at the 3' -end of the gene sequence of RFV-Fd0 (the 4965-6758 interval of NCBI sequence No. NC_ 005084.2); RFV-RBPd21, RFV-RBPd32, RFV-RBPd60 and RFV-RBPd80 are respectively 21, 32, 60 or 80 amino acids truncated after the N end of the peptide chain of RFV-RBPd0 (NCBI serial number is NP-899660.1) starts amino acid M; the gene sequences of RFV-RBP21, RFV-RBPd32, RFV-RBPd60 and RFV-RBPd80 are respectively shortened by 63, 96, 180 or 240 nucleotides after the start codon ATG at the 5' end of the gene sequence of RFV-RBPd0 (the 6762-8699 interval of NCBI serial number NC_ 005084.2). In the embodiment, the effect of RFV is firstly compared with that of the existing NIV and MEV envelope proteins, then the RFV envelope proteins are truncated and optimized, and compared with NIV-Fd22/RBPd34, RFV-Fd0, RFV-Fd13, RFV-Fd32, RFV-Fd56 and RFV-Fd80 are further screened, and the combination with good effect in 25 groups of RFV-RBPd0, RFV-RBPd21, RFV-RBPd32, RFV-RBPd60 and RFV-RBPd80 which are matched with each other is further screened, and the specificity is tested through different cell populations, so that the optimal envelope protein coding region sequence is screened. The sequence used in the invention is shown in a sequence table. The sequence of NIV-Fd22/RBPd34 is derived from Bender R, anke M, schneider I C, et al Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment [ J ]. PLoS Pathogens, 2016, 12 (6): e 1005641. d0 represents no truncation, i.e.wild type, e.g.RFV-Fd 0, i.e.wild type RFV-F, RFV-RBPd0, i.e.wild type RFV-RBP.
The lentiviral envelope protein backbone vector used in this example was pMD2.G (Addgene, accession number: 12259), which was linearized by selection of restriction enzymes PmlI (New England Biolabs, R0532L) and PstI (New England Biolabs, R3140M), and coding regions RFV-F and RFV-RBP-scFv sequences and truncate sequences were amplified respectively by designing Gibson assembly primers based on the linearized vector end sequences. Wherein the scFv-CD34 amino acid and gene sequence are derived from: patent WO2009079922, corresponding nucleic acid sequence EP2233501; the scFv-CD105 amino acid and gene sequence are derived from: anliker B, abel T, kneissl S, et al Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors [ J ]. Nature Methods, 2010, 7 (11): 929-935. Then 0.2 pmol of amplified product was mixed with 0.05 pmol of linearized vector and the total volume was made up to 10. Mu.l using deionized water, 10. Mu.l of 2X Gibson Assembly Master Mix (New England Biolabs, E2611L) was added, mixed well, incubated at 50℃for 1 hour, immediately cooled on ice, and heat shock transformed using E.coli Stbl3 competent cells, cultured overnight at 37℃on ampicillin-containing solid LB plates, single clones were selected for colony PCR identification, positive colonies were subjected to first generation sequencing verification, plasmid unified expansion culture and plasmid extraction with correct sequencing. A representative envelope protein plasmid in the recombinant lentiviral vector obtained by successful construction is shown in FIG. 3. All the sequence synthesis and sequencing work of the viral protein coding region and the corresponding primers are completed by Beijing Liuhua big gene technology Co.
EXAMPLE 2 preparation of recombinant lentiviral vector
The recombinant lentiviral vector of this example co-transfects HEK293T cells from an envelope protein plasmid and a third generation lentiviral packaging plasmid system. The specific operation is as follows:
HEK293T cell density was first adjusted to 4X 10 using DMEM high-sugar medium 5 Uniformly inoculating 2 ml cell suspension into six-hole culture plate, placing at 37deg.C and 5% CO 2 The culture was carried out in an incubator at 24 h to a confluence of about 80%. Wherein the DMEM high-sugar culture medium comprises 10% of fetal bovine serum, 1% of diabody, 1% of L-glutamine solution and 1% of nonessential amino acid solution. As shown in FIG. 2, the plasmid total system of the recombinant lentiviral vector used 4. Mu.g, including helper plasmid psPAX2 (Addgene #12260, https:// www.addgene.org/12260 /), shuttle plasmid pCDH #Addgene #104834, https:// www.addgene.org/104834/, GFP-validating gene inserted within LTR sequence, envelope plasmid pF, envelope plasmid pRBP-scFv, ratio 4:4:3:1, HEK293T,48 h were co-transfected with transfection reagent DNA Transfection Reagent (POLYPLUS, CPT 117), cell culture supernatant was collected and filtered through 0.45 μm filter to remove cell pellet, mixed with virus concentrate Lenti-X Concentrator (TAKARA, 631232), centrifuged at 15000 g for 1 h, supernatant removed, resuspended as virus stock using 100 μl medium, and appropriate split-filled for long term storage at-80 ℃.
Example 3 evaluation of viral titre
RNA was extracted from 10. Mu.l of the virus stock by means of a viral RNA extraction kit (TIANGEN, DP 315), quantified using a Lenti-X ™ qRT-PCR Titration Kit (CLONTECH, 631235), and the lentiviral Vector Copy Number (VCN) in the sample was absolutely quantified using a gradient diluted virus standard as a standard curve, and the vector copy number multiplied by the stock dilution ratio to obtain the virus titer. The viral titer calculation formula is as follows:
example 4 lentiviral transduction
Target cells (HT 1080-CD 34) were grown using the corresponding MEM high sugar medium + Or HT1080-CD105 + ) Non-target cell (HT 1080) density of 5×10 4 Uniformly inoculating 1 ml cell suspension into twelve-well culture plate, standing at 37deg.C and 5% CO 2 The culture was carried out in an incubator at 24 h to a confluence of about 80%. Removing the original culture medium, washing with DPBS buffer solution twice, diluting virus stock solution with the culture medium according to MOI 10-20, adding transfer promoter Polybrene (SANTA CRUZ, sc-134220) with final concentration of 5 μg/ml, mixing, adding target/non-target cells, placing at 37deg.C, 5% CO 2 After 24-h culture in incubator, the supernatant was removed, DPBS buffer was washed twice, and fresh medium was added for further 48 hours.
EXAMPLE 5 evaluation of expression of target Gene and transduction Positive Rate
The expression of Green Fluorescent Protein (GFP) in transduced cells was observed and photographed by a fluorescence inverted fluorescence microscope (OLYMPUS, IX 73), and the cell transduction positive rate was evaluated by a flow cytometer (Beckmem CytoFLEX). The original culture medium is firstly sucked and removed, the DPBS buffer solution is washed twice to thoroughly remove the original culture medium, and digested for 3 min at 37 ℃ by 0.1 ml pancreatin, then MEM high sugar culture medium is added to stop the digestion, the cells are blown into cell suspension, and the cell density is adjusted to 1 multiplied by 10 5 Transferring 1. 1 ml after each ml into a 1.5 ml centrifuge tube, centrifuging for 500 g and 5 min, discarding the supernatant, and washing with 0.5 ml DPBS buffer for 1 time; and (3) centrifuging for 500 g for 5 min again, re-suspending cells with 0.2 ml of DPBS buffer solution after supernatant is discarded, filtering to the bottom of the flow tube through a cell filter screen, blowing and evenly mixing the cell suspension, and placing the flow tube in a sample loading groove of a flow cytometer for sample loading. The position, size and shape of the gate are adjusted in the FSC/SSC diagram, the target cell population is enclosed, the voltage of the fluorescent channel is regulated, fluorescent signals are collected at proper positions, and data are recorded and stored.
EXAMPLE 6 experimental results
(1) Screening of Paramyxoviral envelope proteins
Firstly, a paramyxovirus envelope protein type used for constructing a recombinant lentiviral vector is screened through a delivery efficiency and packaging efficiency test, GFP expression is shown as A in figure 1, transduction positive rate is shown as B in figure 1, transduction positive rate of RFV-F/RBP is increased to 200%, and virus titer is shown as C in figure 1, and virus titer is increased to 200%.
(2) Recombinant lentiviral vectors
To determine whether RFV is advantageous over existing NIV and MEV envelope pseudotyped lentiviral vectors, viral envelope F and RBP gene sequences and their truncate gene sequences need to be constructed into envelope backbone plasmids. Firstly, using restriction endonucleases PmlI and PstI to linearize a pMD2.G vector and a pMD2.G vector containing scFv, and then respectively inserting an RFV-F gene sequence (4965-6758 interval with NCBI sequence number of NC_ 005084.2) and an RFV-RBP gene sequence (6762-8699 interval with NCBI sequence number of NC_ 005084.2) into the linearized pMD2.G vector and the pMD2.G vector containing scFv through a one-step Gibson assembly reaction, wherein the envelope protein plasmid structure of the recombinant lentiviral vector of the example is shown in figure 3. The envelope protein RFV-F plasmid is mainly composed of the following elements: a cytomegalovirus enhancer (CMV enhancer), a cytomegalovirus promoter (CMV promoter), a human β -globin intron (β -globin intron), a Kozak consensus sequence (Kozak), an envelope protein coding region sequence, a human β -globin polyadenylation signal (β -globin poly a). The envelope protein RFV-RBP-scFv plasmid mainly consists of the following elements: cytomegalovirus enhancer (CMV enhancer), cytomegalovirus promoter (CMV promoter), human β -globin intron (β -globin intron), kozak consensus sequence (Kozak), envelope protein coding region sequences, linker sequences (Linker), single chain antibody sequences (scFv), and human β -globin polyadenylation signal (β -globin poly a).
(3) RFV envelope protein transduction Capacity test
The constructed RFV envelope protein plasmid is used for packaging a lentiviral vector, and the transduction positive rate is quantified through GFP expression so as to detect the gene delivery capacity of the vector. GFP expression is shown in FIG. 4A and transduction positive rate and viral titer are shown in FIG. 4B. The results show that, as in the case of the existing paramyxoviral NIV and MEV envelope protein transduction, the RFV envelope protein pseudotyped lentiviral vector is capable of significantly increasing the target cell HT1080-CD34 + Rather than detecting no transduction events in the target cells HT 1080.
(4) Truncated optimization of envelope protein of reptile paramyxovirus
In order to further optimize the RFV envelope protein, the intracellular region and the transmembrane region of the protein are subjected to truncation optimization, the truncation scheme is shown in figure 5, truncations of different degrees of RFV-F and RFV-RBP-scFv are respectively constructed, and the truncations are combined with each other to screen out a combination with better virus packaging titer and transduction efficiency. GFP expression is shown in FIG. 6, transduction positive rate versus viral titer is shown in FIG. 7, and statistical comparison data for each group of transduction positive rates are shown in tables 2 and 3. The combination of RFV-Fd13/RBPd21 increased to 200% compared to the non-truncated protein virus titer by combining the transduction positive rate and the virus titer, and the transduction positive rate increased to 200%.
Table 2 data for statistically comparing the transduction positive rates of the groups
Table 3 data for statistical comparison of transduction positive rates for each group
(5) Targeted lentiviral vector specificity assessment
To test the specificity of the screened targeted lentiviral vectors, the test was performed using the transduction of the vector on target/non-target cells. GFP expression is shown in FIG. 8A, and transduction positive rate is shown in FIG. 8B. The results showed a 200% increase in the positive rate of RFV-Fd13/RBPd21-scFv-CD34 transduction compared to the NIV truncating pseudotyped lentivirus. At the same time, the RFV-Fd13/RBPd21-scFv-CD105 transduction positive rate was increased to 200% (as shown in FIG. 9A and FIG. 9B). In a mixed cell population with 1:1 ratio of target cells to non-target cells, the RFV-Fd13/RBPd21-scFv can significantly improve the transduction efficiency in the target cells, but no transduction condition is detected in the non-target cells. In a mixed cell population with 1:3 target cells and non-target cells, the transduction efficiency of RFV-Fd13/RBPd21-scFv-CD34 in the target cells can be remarkably improved. Compared to the NIV truncating pseudotyped lentivirus, the RFV-Fd13/RBPd21-scFv-CD34 transduction positive rate was increased by 50%, whereas no significant transduction was detected in the non-target cells (as in FIG. 10A and FIG. 10B).
Claims (28)
1. An envelope protein combination, comprising RFV-F or a variant thereof and RFV-RBP or a variant thereof; the NCBI sequence number of the amino acid sequence of the RFV-F is NP-899659.1; the NCBI sequence number of the amino acid sequence of the RFV-RBP is NP-899660.1; variants of RFV-F are those in which a truncation of 1-80 amino acid residues occurs at RFV-F; a variant of RFV-RBP is a truncation of 1-80 amino acid residues occurring at the RFV-RBP.
2. The envelope protein combination of claim 1, wherein the variant of RFV-F and the variant of RFV-RBP truncate a portion or all of the intracellular region, a portion or all of the transmembrane region and/or a portion of the extracellular region, respectively.
3. The envelope protein combination of claim 2, wherein the amino acid sequence of the variant of RFV-F is a C-terminal truncated 13, 32, 56 or 80 amino acids of the sequence of NCBI sequence No. np_ 899659.1; the amino acid sequence of the variant of RFV-RBP is an amino acid sequence truncated by 21, 32, 60 or 80 amino acids after the N-terminal initial amino acid M of the amino acid sequence of NCBI sequence No. NP-899660.1.
4. The combination of envelope proteins of claim 3, wherein the combination of envelope proteins comprises the amino acid sequence of NCBI sequence No. np_899659.1 and the amino acid sequence of NCBI sequence No. np_ 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence of NCBI sequence No. np_ 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
The envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 32 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 32 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 60 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence with NCBI sequence number NP 899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminal initial amino acid M of the amino acid sequence with NCBI sequence number NP 899660.1; or alternatively, the first and second heat exchangers may be,
The envelope protein combination comprises an amino acid sequence truncated by 32 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 80 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 56 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 32 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence of NCBI sequence No. NP-899659.1 and an amino acid sequence truncated by 60 amino acids after the N-terminal starting amino acid M of the amino acid sequence of NCBI sequence No. NP-899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 60 amino acids after the N-terminal starting amino acid M of the amino acid sequence of NCBI sequence No. np_ 899660.1; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 13 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 80 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
The envelope protein combination comprises an amino acid sequence truncated by 56 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 80 amino acids after the N-terminus of the amino acid sequence of NCBI sequence No. np_899660.1 initiates amino acid M; or alternatively, the first and second heat exchangers may be,
the envelope protein combination comprises an amino acid sequence truncated by 56 amino acids at the C-terminus of the sequence of NCBI sequence No. np_899659.1 and an amino acid sequence truncated by 21 amino acids after the N-terminal starting amino acid M of the amino acid sequence of NCBI sequence No. np_ 899660.1.
5. The envelope protein combination of any one of claims 1-4, wherein the RFV-RBP or variant thereof further comprises an scFv fused thereto.
6. An envelope protein plasmid comprising a nucleotide sequence encoding the envelope protein combination of any one of claims 1-5; wherein the nucleotide sequences of RFV-F or variants thereof and RFV-RBP or variants thereof are located on the same envelope plasmid or on two different envelope plasmids pF and pRBP, respectively.
7. The envelope protein plasmid according to claim 6, wherein the nucleotide sequence of RFV-F is the 4965-6758 interval of NCBI sequence No. nc_ 005084.2; the nucleotide sequence of the RFV-RBP is 6762-8699 interval with NCBI sequence number NC_ 005084.2.
8. The envelope protein plasmid of claim 7, wherein the nucleotide sequence of the variant of RFV-F is a nucleotide sequence truncated by 39, 96, 168 or 240 nucleotides at the 3' end of the sequence in the 4965-6758 interval of NCBI sequence No. NC 005084.2; the nucleotide sequence of the variant of RFV-RBP is truncated by 63, 96, 180 or 240 nucleotides after the 5' -terminal initiation codon ATG of the sequence of interval 6762-8699 of NCBI sequence No. NC_ 005084.2.
9. The envelope protein plasmid of claim 8, wherein the nucleotide sequences encoding the envelope protein combination comprise the nucleotide sequence of the 4965-6758 interval of NCBI sequence No. nc_005084.2 and the nucleotide sequence of the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 39 nucleotides at the 3' end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a nucleotide sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence 3 'to the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 63 nucleotide truncated nucleotide sequence after the 5' start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
The nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 96 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 63 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 96 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 180 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
The nucleotide sequence encoding the envelope protein combination comprises the nucleotide sequence of the 4965-6758 interval of NCBI sequence No. NC_005084.2 and the nucleotide sequence truncated by 63 nucleotides after the 5' -terminal start codon ATG of the sequence of the 6762-8699 interval of NCBI sequence No. NC_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 96 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 240 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 168 nucleotides at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a nucleotide sequence truncated by 96 nucleotides after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises the nucleotide sequence of the 4965-6758 interval of NCBI sequence No. NC_005084.2 and the nucleotide sequence truncated 180 nucleotides after the 5' -terminal start codon ATG of the sequence of the 6762-8699 interval of NCBI sequence No. NC_ 005084.2; or alternatively, the first and second heat exchangers may be,
The nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 180 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a 39 nucleotide truncated nucleotide sequence at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a 240 nucleotide truncated nucleotide sequence after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 168 nucleotides at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. nc_005084.2 and a nucleotide sequence truncated by 240 nucleotides after the 5' end start codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. nc_ 005084.2; or alternatively, the first and second heat exchangers may be,
the nucleotide sequence encoding the envelope protein combination comprises a nucleotide sequence truncated by 168 nucleotides at the 3 'end of the sequence in the 4965-6758 interval of NCBI sequence No. NC_005084.2 and a nucleotide sequence truncated by 63 nucleotides after the 5' end initiation codon ATG of the sequence in the 6762-8699 interval of NCBI sequence No. NC_ 005084.2.
10. The envelope protein plasmid of claim 6, wherein the RFV-RBP or variant thereof further comprises an scFv fused thereto.
11. The envelope protein plasmid according to any one of claims 6 to 10, further comprising the following genetic elements: cytomegalovirus enhancer, cytomegalovirus promoter, human β -globin intron, kozak consensus sequence, and/or human β -globin polyadenylation signal.
12. The envelope protein plasmid of claim 11, wherein the backbone vector employed by the envelope protein plasmid is a pmd2.G vector.
13. A lentiviral vector packaging system comprising the envelope protein plasmid of any one of claims 6-12.
14. The lentiviral vector packaging system of claim 13, further comprising a helper packaging plasmid and/or a shuttle vector plasmid.
15. The lentiviral vector packaging system of claim 14, wherein the ratio of the helper packaging plasmid, the shuttle vector plasmid, and the envelope protein plasmid is (3-1): 3-1.
16. The lentiviral vector packaging system of claim 15, wherein the ratio of the helper packaging plasmid, the shuttle vector plasmid, and the envelope protein plasmid is 1:1:1.
17. The lentiviral vector packaging system of claim 16, wherein the ratio of envelope protein plasmids pF and pRBP is (1-5): 1; the helper packaging plasmid is psPAX2 and the shuttle vector plasmid is pCDH.
18. The lentiviral vector packaging system of claim 17, wherein the ratio of envelope protein plasmids pF and pRBP is 3:1.
19. A transformant transfected with an envelope protein plasmid according to any one of claims 6 to 12 or a lentiviral vector packaging system according to any one of claims 13 to 18.
20. The transformant of claim 19, wherein the recipient cell of the transformant is an animal cell.
21. The transformant of claim 20, wherein the recipient cell of the transformant is HEK293T.
22. A targeted viral vector comprising the combination of envelope proteins according to any one of claims 1-5.
23. The targeted viral vector of claim 22, wherein the targeted viral vector is obtained by culturing the transformant of any one of claims 19 to 21.
24. A method for preparing the targeted viral vector of claim 22 or 23, wherein the targeted viral vector is obtained by culturing the transformant of any one of claims 19 to 21.
25. A pharmaceutical composition comprising the transformant of any one of claims 19 to 21 or the targeted viral vector of claim 22 or 23.
26. A kit comprising the envelope protein plasmid of any one of claims 6 to 12, the lentiviral vector packaging system of any one of claims 13 to 18, the transformant of any one of claims 19 to 21, the targeted viral vector of claim 22 or 23, or the pharmaceutical composition of claim 25.
27. A method of delivering a gene, ribonucleoprotein complex or drug, characterized in that the method is for gene, ribonucleoprotein complex or drug delivery by an envelope protein combination according to any one of claims 1-5, an envelope protein plasmid according to any one of claims 6-12, a lentiviral vector packaging system according to any one of claims 13-18, a transformant according to any one of claims 19-21, a targeted viral vector according to claim 22 or 23, a pharmaceutical composition according to claim 25 or a kit according to claim 26; the method is for non-therapeutic purposes.
28. Use of an envelope protein combination according to any one of claims 1 to 5, an envelope protein plasmid according to any one of claims 6 to 12, a lentiviral vector packaging system according to any one of claims 13 to 18, a transformant according to any one of claims 19 to 21, a targeted viral vector according to claim 22 or 23, a pharmaceutical composition according to claim 25 or a kit according to claim 26 for the preparation of a gene therapy drug.
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